Nucleic acid bulge-detecting agent

ABSTRACT

This invention relates to a metal complex of formula (I). The metal complex is capable of cleaving a bulge-containing nucleic acid at the bulge site with high specificity.

BACKGROUND OF THE INVENTION

[0001] Nucleic acid bulges refer to regions of unpaired bases in adouble-stranded nucleic acid molecule. These bulges have been known totake part in many important biological processes.

[0002] For example, ENA bulges form crucial motifs for specific nucleicacid-protein recognition and binding. It has been known that the humanimmunodeficiency virus transactivator protein Tat binds to athree-pyrimidine bulge in the response element TAR. See, e.g., Weeks etal., Science 249, 1281-1285 (1990). Nucleic acid bulges also produceframeshift mutations which can change the product of the proteintranslation and result in various disorders. According to one report,Myerowitz et al., J. Biol. Chem. 263, 18587-18589 (1988), approximately70% of Ashkenazi Tay-Sachs disease is caused by a four-base pairinsertion mutation in the HEX A gene encoding the α-subunit ofhexosaminidase A. Another disease, cystic fibrosis, is also caused byframeshift mutation. A three-base pair deletion (AF508) is commonlyfound among cystic fibrosis patients. Rommens et al., Am. J. Hum. Genet.46, 395-396 (1990).

[0003] Comparative gel electrophoresis assay has been used to detect-thepresence of bulges in nucleic acids. This assay differentiates nucleicacids with and without bulges by their different mobility in gel.However, it can only provide information as to whether a nucleic acidcontains a bulge. Thus, there exists a need for a detection method whichcan provide additional information, e.g., the location of a bulge in anucleic acid.

SUMMARY OF THE INVENTION

[0004] One aspect of this invention relates to a metal complex offormula (I):

[0005] Each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸, independently, ishydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl,amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl,alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy,cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl. Each of R² and R³, and R⁶ and R⁷, independently,optionally join together to form a cyclic moiety which is fused with thetwo pyridyl rings to which R² and R³, or R⁶ and R⁷ are bonded. Thecyclic moiety, if present, is optionally substituted with alkyl, alkoxy,hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl,alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl,alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl,heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each ofL¹ and L², independently, is —C(R^(a)) (R^(b))—, —O—, —S—, or —N(R^(c))—and each of R^(a), R^(b), and R^(c), independently, is hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl. M is a Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re,Ir, Pt, or Pd ion, and the oxidation state of M is 0, 1, 2, 3, or 4.Each of X¹ and X², independently, is a labile ligand.

[0006] Examples of a metal complex of formula (I) includecobalt(II)(hexaazacyclophane)(trifluoroacetate)₂, cobalt (II)(hexaazacyclophane) (H₂O) (trifluoroacetate) ruthenium(II)(hexaazacyclophane) (trifluoroacetate)₂, andmanganese(II)(hexaazacyclophane)(trifluoroacetate)₂.

[0007] Another aspect of this invention relates to a method ofspecifically cleaving a nucleic acid bulge. The method comprisingcontacting the bulge with a metal complex of formula (I), supra, where Mis a Fe, Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re, Ir, Pt, or Pdion. In one embodiment, the method is performed in the presence of anoxidant, e.g., hydrogen peroxide, or in a medium having a pH valueswhich ranges from 4-9.

[0008] In this disclosure, a nucleic acid bulge is a region in adouble-stranded nucleic acid molecule (DNA or RNA), the region having atleast one unpaired nucleotide and being flanked by two pairednucleotides. The nucleic acid bulge can contain 1-5 unpaired nucleotides(e.g., 1-3). Using nucleic acid substrate A in FIG. 1 as an example, thenucleic acid bulge present therein contains three unpaired nucleotides,i.e., T₆, C₇, and T₈. This three-base bulge is flanked by two pairednucleotides, i.e., A₅-T₂₃ and G₉-C₂₂. In contrast, the C₁₃-A₁₈ hairpinloop, which is also present in substrate A, is not a bulge as theunpaired nucleotides are only connected to one paired nucleotide, i.e.,C₁₂-G₁₉. A nucleic acid bulge can also contain two nucleotides. See thebulge present in substrate D which is formed of two unpairednucleotides, i.e., C. and T₇.

[0009] A salt of a metal complex of formula (I) is also within the scopeof this invention. Note that a metal complex of formula (I) can bepositively charged. A salt of such a metal complex can be formed with ananionic counterion. Examples of counterions include fluoride, chloride,bromide, iodide, sulfate, sulfite, phosphate, acetate, oxalate, andsuccinate.

[0010] As described above, each of R² and R³, and R⁶ and R⁷,independently, can join together to form a cyclic moiety. The cyclicmoiety can contain 5 or 6 ring members and can be cycloalkyl,heterocycloalkyl, aryl, or heteroaryl. For example, when the cyclicmoiety formed by joining R² and R³ is a benzene, it fuses with the twopyridine rings co which R² and R³ are bonded, and the benzene ring andthe two pyridine rings together form phenanthroline.

[0011] As used herein, alkyl is a straight or branched hydrocarbon chaincontaining 1 to 6 carbon atoms. Examples of alkyl include, but are notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, and hexyl.

[0012] By “cycloalkyl” is meant a cyclic alkyl group containing 3 to 8carbon atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl,cyclohexyl, cycloheptyl, adamantyl, and norbornyl. Heterocycloalkyl is acycloalkyl group containing 1-3 heteroatoms such as nitrogen, oxygen, orsulfur. Examples of heterocycloalkyl include piperidine, piperazine,tetrahydropyran, tetrahydrofuran, and morpholine.

[0013] In this disclosure, aryl is an aromatic group containing 6-12ring atoms and can contain fused rings, which may be saturated,unsaturated, or aromatic. Examples of an aryl group include phenyl andnaphthyl. Heteroaryl is aryl containing 1-3 heteroatoms such asnitrogen, oxygen, or sulfur. Examples of heteroaryl include pyridyl,furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,benzofuranyl, and benzothiazolyl.

[0014] Note that an amino group can be unsubstituted, mono-substituted,or di-substituted. It can be substituted with groups such as alkyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Halo refers tofluoro, chloro, bromo, or iodo.

[0015] A labile ligand (i.e., X¹ or X²) refers to a group whichcoordinates with less affinity to the metal ion (i.e., M) of a complexof formula (I) relative to the four pyridyl nitrogen atoms. Such ligandcan therefore undergo rapid equilibrium with other labile ligands.Examples of a labile ligand include H₂O, Cl, trifluoroacetate, orpyridine.

[0016] A metal complex of formula (I) possesses unexpectedly highspecificity toward nucleic acid containing a bulge structure. Asdescribed above, a nucleic acid with such a structure is associated withvarious disorders. A metal complex of formula (I) can therefore be usedin a diagnostic kit for detecting nucleic acid bulge-associateddisorders.

[0017] Other features or advantages of the present invention will beapparent from the following detailed description of several embodiments,and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWING

[0018]FIG. 1 depicts the sequence of each of nucleic acid substrates A-Dused in Examples 1-3 below.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The invention features metal complexes of formula (I) whichspecifically target bulge structures in a nucleic acid molecule Metalcomplexes of formula (I) can therefore be used in detecting nucleic acidbulges for diagnostic purposes. A method of specifically cleaving anucleic acid bulge using a metal complex of formula (I) is also withinthe scope of this invention.

[0020] A number of methods can be used to prepare the metal complexes offormula (I). For example, when each of L¹ and L² is —N(R^(c))— whereR^(c) is H, the hexaazacyclophane car be formed by reacting2,9-diamino-1,10-phenanthroline with 2,9-dichloro-1,10-phenanthrolinethe presence of nickel (II) ion, which can then be removed by usingtrifluoroacetic acid. See, e.g., Chang et al., J. Chin. Chem. 43, 73-75(1996).

[0021] Alternatively the method described above can be modified as shownin steps i, ii, and iii of the following scheme:

[0022] Reagents and conditions i, CH₃L K₃Fe(CN)₆/NaOH(aq); ii, PCl₅,POCl₃, 75% iii, NH_(3(g)), 200° C. 80%, iv, Co(OAc)₂ in TFA/MeOH/CH₂Cl₂,reflux, 64%.

[0023] As shown in step iv above, the desired metal ion, e.g.,cobalt(II), can be coordinated to the hexaazacyclophane ligand at thesame time as two axial ligands, e.g., trifluoroacetate, are coordinatedto the metal ion. Substituents on the aromatic rings can be introducedeither before or after the preparation of the ligand by methods familiarto one of ordinary skill in the art, e.g., electrophilic aromaticsubstitution.

[0024] Complexes of formula (I) where each of L¹ and L² is —S— can beprepared in an analogous way by reacting starting materials such as2,9-dichloro-1,10-phenanthroline in H₂S gas instead of ammonia gas (seestep iii of the above scheme). On the other hand, a complex of formula(I) where each of L¹ and L² is —O— or —C(R^(a)) (R^(b))— can be preparedby reacting compounds such as 1,10-phenanthroline-2,9-diboronic acid and2,9-dihydroxy-1,10-phenanthroline in the presence of a palladiumcatalyst such as Pd(PPh₃)₄.

[0025] Note that the metal ion of each of the complexes of formula (I)adopts an octehedral coordination. For example, the X-ray crystalstructure of cobalt(II)(hexaazacyclophane)(trifluoroacetate) 2 i.e.,Co^(II)(HAPP)(TFA)₂, reveals that the complex contains two labile axialTFA ligands, and two linked 1,10-phenanthroline moieties where all fourpyridyl nitrogen atoms are locating on the same coordination plane. Theaverage Co—N distance is approximately 1.86 Å. EPR spectrum of theCo^(II) complex gave a g_(av) value at 2.005-2.331 in methanol,indicating the presence of an octahedral Co^(II) complex. When oneequivalent of pyridine was added, it rapidly displaced one of the axialTFA ligands under ambient conditions, as monitored by EPR spectroscopy,suggesting that the TFA ligands are labile. The TFA ligands can also bereadily substituted by water upon dissolution of the complex in aqueousbuffer.

[0026] Due to the steric hindrance brought about by its octahedralstructure, a metal complex of formula (I) does not intercalate inbetween bases of a nucleic acid molecule. Using Co^(II)(HAPP) (TFA)₂ asan example, a topoisomerase I assay conducted in the absence of H₂O₂ andunder non-cleavage conditions (vide infra) showed no sign of DNAunwinding resulted from DNA intercalation. Further, under the samenon-cleavage conditions, a native gel mobility shift assay conductedusing the Co^(II) complex also showed no indication of the presence ofhigh-molecular-weight bands attributable to the presence of aDNA-Co^(II) complex adduct in polyacrylamide gel electrophoresis. Inaddition, the melting temperature of calf thymus DNA (60 μM pernucleotide) incubated with the Co^(II) complex (8 μM) only changed by0.5-1.0° C. In contrast, incubation of DNA with ethidium bromide, aknown DNA intercalator, resulted in a DNA-ethidium bromide adduct with amelting temperature differing by 12-13° C. from the control underidentical reaction conditions. Moreover, the DNA-binding constant of theCo^(II) complex, as determined by spectral titration at 399 nm with calfthymus DNA was found to be 10-fold less when compared to another knownDNA-intercalator, Cu^(II)(HAPP)⁺², which adopts a planar structure.Because of its non-intercalating nature, a metal complex of formula (I)can unambiguously detect the location of a bulge in a nucleic acid.

[0027] In the presence of H₂O₂ (0.005%-0.05%), a metal complex offormula (I) cleaves a nucleic acid molecule containing a bulgecatalytically. Although the metal complex can still effect nucleic acidcleavage in the absence of H₂O₂, a longer reaction time (about 8-10times longer) and a higher concentration of the metal complex (about10-fold higher) are required to produce the same amount of cleavage.When H₂O₂ is replaced by magnesium monoperoxyphthaiic acid or oxone, nosignificant nucleic acid cleavage was observed under the same reactionconditions and time. Further, the amount of nucleic acid cleavage wasreduced by half when a hydroxyl radical scavenger was added. See Example1 below. This indicates that hydroxyl radicals are responsible for thenucleic acid cleavage.

[0028] Moreover, the metal complex targets nucleic acid with highspecificity. Not only does the complex cleave specifically at the bulgestructure, the size of such a structure also controls the specificity ofthe cleavage reaction. It was unexpectedly found that in treating adouble-stranded DNA substrate containing a three-base bulge and asix-base hairpin loop with Co^(II)(HAPP) (TFA)₂, cleavage occurredspecifically at the bulge, and only weakly at the loop. See Example 1below. As hydroxyl radicals are diffusible and generally lackspecificity towards a particular nucleotide or a group of nucleotides,the high specificity must have resulted from a specific recognitionbetween the metal complex and the bulge structure. Indeed, when thejust-described nucleic acid was denatured, no specific cleavage wasobserved at the sequence corresponding to the bulge. See Example 2below.

[0029] Without further elaboration, it is believed that one of ordinaryskill in the art can, based on the description herein, utilized parts orthe whole procedure to its full extent. The following examples are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. All publicationsmentioned above are incorporated by reference in their entirety.

EXAMPLE 1

[0030] λ-Phage FC-174 DNA was purchased from Life Technologies (GibcoBRL). No further purification was needed prior to use. The synthetic DNAsubstrate employed herein was a 27-mer DNA,5′-GCACATCTGAGCCTGGGAGCTCTCTGC-3′ (SEQ ID No. 1) which was purchasedfrom Perkin Elmer Inc. (see nucleic acid substrate A in FIG. 1), and waspurified by gel purification in a 20% denaturing polyacrylamide gel (7 Murea). The DNA bands were visualized with an UV lamp (λ_(max)=254 nm) byplacing the gel on a TLC F254 plate (20×20 cm, Merck). After asuccessive process of excising the desired visible bands, extracting theDNA from gel, and precipitating it by EtOH, a pure DNA was obtained. TheDNA concentration was determined using the extinction coefficient(λ_(max)=260 nm) or molecular weight method (1 OD=about 33 mg and theaverage molecular weight of one nucleotide=330 daltons).

[0031] The 5′-³²P-end labeled DNA substrate was prepared by using T4polynucleotide kinase (New England Biolabs) anddeoxyadenosine-5′-[γ-³²P]-triphosphate (Amersham). The excess freeγ-³²P-ATP was removed by filtration with Centricon-10 (Amicon) usingultracentrifuge (6,000 rpm, Beckman GS-15R equipped with rotor F0850) at4° C. for 80 minutes, followed by an additional centrifuge with Milli-Qwater (1 mL) for 60 minutes. A further dilution to proper radiationintensity with deionized water was performed prior to use in assaysdescribed below.

[0032] Using the 5′-³²P-end labeled DNA substrate, a modifiedMaxam-Gilbert G Lane was prepared by cooling a 20 μL solution containingabout 10 nCi ³²P-labeled substrate in deionized H₂O to 0-4° C. prior tothe addition of dimethyl sulfate. The solution containing the labeledDNA was then vortexed (<1 sec), and 2-mercaptoethanol (10 μL) wasimmediatelyadded to the solution. The solution was vortexed for anadditional 30 seconds. Afteradding to sonicated calf thymus DNA (5 mg)and 3.0 M sodium acetate (pH 7.0, 15 μL) to the solution, the labeledDNA was precipitated with 95% EtOH and centrifuged to obtain a pelletwhich was then treated with piperidine as described above prior to useas control in a DNA cleavage assay.

[0033] In the DNA cleavage assay, a 20 μL solution containing a finalconcentration of about 8 nCi of 5′-γ-³²P-labeled substrate (4-5 μM) andunlabeled DNA (4 mM) in 10 mM sodium phosphate buffer (pH 6.96) werecombined with Co^(II)(HAPP) (TFA)₂ (0.6 μM) and H₂O₂ (0.005-0.05%) at25° C. for 5 minutes. The reaction was quenched by adding sonicated calfthymus DNA (4 mg), 3 M sodium acetate (5 μL, pH 4.5), and 95% EtOH (700μL), and then stored at −78° C. for 20 minutes, centrifuged (12,000 rpm)at 4° C. for 20 minutes, and finally lyophilized to dryness to form apellet. The reaction mixture was then subjected to a piperidinetreatment by adding 0.7 M piperidine aqueous solution (60 μL) andheating at 90° C. for 30 minutes. After the reaction mixture waslyophilized, washed with deionized H₂O, and lyophilized again todryness, it was resuspended in a gel-loading buffer (5 μL) containing0.25% bromophenol blue, 0.25% xylene cyanol FF, and 7 M urea. The DNAfragment was analyzed by 20% denaturing polyacrylamide gel (7 M urea)and then visualized using Kodak BioMax MR-1 films with intensifyingscreens. The optical density of DNA fragments was quantified using imageprograms from NIH image (free shareware) and UVP Inc.(GelBase/GelBlotTMPro) equipped with an Vista S-12 scanner (UMAX).

[0034] The DNA substrate employed in this example contains a three-basebulge and a six-base hairpin loop (see nucleic acid substrate A in FIG.1). This DNA sequence was designed based on the RNA hairpin from thetrans-activation response element (TAR-RNA). After piperidinetreatment,the strand scission was unexpectedly found to occur specifically at thebulge (T₆, C₇, and T₈) and only very weakly at the hairpin loop(C₁₃-A₁₈). Note that both the bulge and the loop contain the same5′-CTG-3′ sequence. Minor cleavage was also found at the sites near theflanking junctions of these nucleotides. Further, no significantoxidative cleavage was observed at the 5′-GGG-3′ region in the DNAhairpin loop which have been reported to be susceptible to oxidativecleavage due to its low reduction potential. When Pt(terpy) (HET)⁺(HET=2-hydroxyethylenethiol), a known DNA intercalator which targets DNAbulges, was added to the reaction, competitive inhibition was observedand the amount of cleavage at the bulge was found to reduce remarkably.

[0035] In the absence of H₂O₂, the reaction required a higherconcentration of the Co^(II) complex (>50 μM) as well as a longerreaction time (>40 minutes) to afford the same amount of DNA cleavage atthe bulge. Moreover, when magnesium monoperoxyphthalic acid (MMPP) andoxone (KHSO,) were used instead of H₂O₂, no significant DNA cleavage wasobserved. Since the addition of superoxide dismutase and D₂O into thereaction medium did not reduce the concentration of circular DNA (FormII) formed in the DNA cleavage products mediated by this Co^(II)complex, superoxide and singlet oxygen species are not involved in thisprocess. Further, when mannitol, a hydroxyl radical scavenger, was addedinto the DNA cleavage assay medium, the amount of circular DNA (Form II)was found to be reduced by half.

[0036] The results described above showed that (1) Co^(II) (HAPP) (TFA)₂specifically cleaves DNA bulge, and (2) the cleavage reaction iseffected by hydroxyl radicals produced by the reaction of the Co^(II)complex with H₂O₂.

EXAMPLE 2

[0037] A 26-mer (D, FIG. 1) was used as the DNA substrate. It wasprepared according to the same procedures as described in Example 1.Note that substrate D only differs from substrate A in that its bulgecontains one less base.

[0038] Co^(II)(HAPP) (TFA)₂ (0.6 μM) was added to substrate D under thesame cleavage reaction conditions as described in Example 1 above.Enhanced and specific cleavage activity was observed at T₇ (in the bulgeregion). The cleavage was found to be inhibited by Pt(terpy)(HET)⁺.

EXAMPLE 3

[0039] Co^(II)(HAPP) (TFA)₂ (0.6 μM) was allowed to react underidentical conditions as described above with a single-stranded 16-mer ofthe sequence 5′-GCCACATCTGAGCCTG-3′ (SEQ ID No. 2) (B, FIG. 1) in thepresence of H₂O₂. No specific cleavage was observed at the 5′-TCT-3′site, even when the concentration of the cobalt complex was increased by20-fold. The single-stranded substrate was then allowed to anneal with acomplementary DNA strand to form a double-stranded DNA with a three-basebulge (C, FIG. 1). When the Co^(II) complex was added to thedouble-stranded substrate, enhanced DNA cleavage was observed at the5′-TCT-3′ bulge. These results indicate that the Co^(II) complex servesas a DNA bulge-specific cleavage reagent without significant specificitytowards the corresponding sequence in the single-stranded DNA.

Other Embodiments

[0040] From the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. For example, a metal complex of formula (I) can be usedto effect cleavage at a nucleic acid substrate with a hairpin loop of1-5 bases. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A metal complex of the following formula:

wherein each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, independently, ishydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl,amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl,alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy,cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl; each of R² and R³, and R⁶ and R³, independently,optionally joining together to form a cyclic moiety fused with the twopyridyl rings to which R² and R³, or R⁶ and R⁷ are bonded; the cyclicmoiety, if present, optionally being substituted with alkyl, alkoxy,hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl,alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl,alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl,heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; each ofL¹ and L², independently, is —C(R^(a)) (R^(b))—, —O—, —S—, or—N(R^(c))—; each of R^(a), R^(b), and R^(c), independently, is hydrogen,alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; M is a Co, Ni, Ru, Rh, Mn, Os, Ag, Cr. Zn, Cd, Hg, Re,Ir, Pt, or Pd ion; and each of X¹ and X², independently, is a labileligand; or a salt thereof.
 2. The metal complex of claim 1, wherein eachof R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, independently, is hydrogen,alkyl, or alkoxy.
 3. The metal complex of claim 1, wherein each of R²and R³, and R⁶ and R⁷, independently, join together to form a cyclicmoiety; the cyclic moiety being benzene.
 4. The metal complex of claim3, wherein the cyclic moiety is unsubstituted.
 5. The metal complex ofclaim 4, wherein each of R¹, R⁴, R⁵, and R⁸, independently, is hydrogen,alkyl, or alkoxy.
 6. The metal complex of claim 5, wherein each ofR^(, R) ⁴, R⁵, and R⁸, independently, is hydrogen.
 7. The metal complexof claim 6, wherein each of L¹ and L², independently, is —N(R^(c))—where R^(c) is hydrogen.
 8. The metal complex of claim 7, wherein M isCo.
 9. The metal complex of claim 8, wherein X¹ and X², independently,is trifluoroacetate.
 10. The metal complex of claim 9, wherein saidcomplex is cobalt(II)(hexaazacyclophane)(trifluoroacetate)₂.
 11. Themetal complex of claim 1, wherein each of L¹ and L², independently, is—S— or N(R^(c))—.
 12. The metal complex of claim 11, wherein each of L¹and L², independently, is —N(R^(c))— where R^(c) is hydrogen.
 13. Themetal complex of claim 1, wherein M is Co, Ru, or Mn.
 14. The metalcomplex of claim 13, wherein M is Co.
 15. The metal complex of claim 1,wherein X¹ and X², independently, is H₂O, Cl, trifluoroacetate, orpyridine.
 16. The metal complex of claim 15, wherein X¹ and X²,independently, is trifluoroacetate.
 17. A method of specificallycleaving a nucleic acid bulge, the method comprising contacting thenucleic acid bulge with a metal complex having the following formula:

wherein each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, independently, ishydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl,amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl,alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy,cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl; each of R² and R³, and R⁴ and R⁵, independently,optionally joining together to form cyclic moiety fused with the twopyridyl rings to which R² and R³, or R⁴ and R⁵ are bonded; the cyclicmoiety, if present, optionally being substituted with alkyl, alkoxy,hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl,alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl,alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl,heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; each ofL¹ and L², independently, is —C(R^(a))(R^(b)), —O—, —S—, or —N(R^(c))—;each of R^(a), R^(b), and R^(c), independently, is hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; M is a Fe, Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg,Re, Ir, Pt, or Pd ion; and each of X¹ and X², independently, is a labileligand; or a salt thereof.
 18. The method of claim 17, wherein each ofR² and R³, and R⁶ and R⁷, independently, join together to form a cyclicmoiety; the cyclic moiety being benzene.
 19. The method of claim 18,wherein the cyclic moiety is unsubstituted.
 20. The method of claim 19,wherein each of R¹, R⁴, R⁵, and R⁸, independently, is hydrogen.
 21. Themethod of claim 20, wherein each of L¹ and L², independently, is—N(R^(c))— where R^(c) is hydrogen.
 22. The method of claim 21, whereinM is Co.
 23. The method of claim 22, wherein X¹ and X², independently,is trifluoroacetate.
 24. The method of claim 23, wherein said complex iscobalt(II) (hexaazacyclophane) (trifluoroacetate)₂.
 25. The method ofclaim 17, wherein the method is performed in the presence of hydrogenperoxide.
 26. The method of claim 17, wherein the nucleic acid bulgecontains 1-5 unpaired nucleotides.
 27. The method of claim 26, whereinthe nucleic acid bulge contains 1-3 unpaired nucleotides.